This invention relates generally to the immunoglobulins, and more particularly to a cell surface molecule-induced macrophage activation that results in the rejection by the host of the cell expressing the cell surface molecule.
Cell therapy seeks to provide biologically active molecules to a patient by implanting cells that produce the biologically active molecules into the patient. In unencapsulated cell therapy approaches, xe2x80x9cnakedxe2x80x9d cells are implanted. Several approaches have been taken to prevent rejection of the implanted xe2x80x9cnakedxe2x80x9d cells. Treatments include immunosuppression of the patient, pre-clearance from the recipient serum of natural antibodies, or administration of high doses of low molecular weight haptens to inhibit natural antibody binding to transplanted tissue. Alternatively, researchers have proposed the alteration of cells to reduce or eliminate expression of antigen or epitopes that stimulate rejection of the cells or tissue by natural antibodies in a recipient. However, using mitotically active cells creates a risk of tumorigenicity of the implanted tissue.
In encapsulated cell therapy approaches, a permselective physical barrier immunoisolates the implanted tissue from the host tissue. The barrier permits passage of the desired molecules between the patient and the encapsulated tissue, but protects the encapsulated cells or tissue from destruction by the immune system of the patient. Use of xenogeneic tissue or cells in encapsulated cell therapy also acts as a xe2x80x9csafety feature,xe2x80x9d because encapsulated cells are rejected by the patient""s immune system if the capsule breaks or ruptures.
A patient""s immune system has several components, some of which are useful for encapsulated cell therapy and some of which are undesirable. In one component, phagocytes scavenge target cells, such as the xenogeneic cells described above In particular, antibody-dependent cell-mediated cytotoxicity (ADCC) has an important role in the destruction of many target cells, including tumor cells, by macrophages. Opsonization of target cells with immunoglobulin G (IgG) enhances the removal of these materials from a host. The role of macrophages in the destruction of target cells by ADCC in the presence of specific antibodies has been well established. While the selectivity of macrophage targeting is based on antibody specificity, the lytic attack on the target cells is triggered by Fc receptor-mediated ADCC.
Another component of the immune system is the activation of the complement system. The two pathways of complement activation (the classical and the alternative pathways) are both directed at a central step in complement activation, the cleavage of C3. A single terminal pathway is the formation of a membrane attack complex (MAC). The classical pathway is normally activated by antigen-antibody complexes., where certain antibodies are complement fixing (capable of binding to complement to cause activation of the classical pathway). Activation of the classical pathway can be initiated with binding of C1q, the first factor of complement cascade, to the Fc region of immunoglobulin. Then, a cascade of proteolytic events results in the activation of C5 convertase, which cleaves C5 into C5b and C5a. The C5b then binds C6, C7, C8 to form a C5b-8 complex. Binding of C9 molecules to C5b-8 forms C5b-9 (the MAC), which inserts into lipid bilayers and forms transmembrane channels that permit bidirectional flow of ions and macromolecules. By this mechanism, complement causes lysis of the cells.
The complement system is important in host defense, but activation at inappropriate sites or to an excessive degree can cause host tissue damage. Complement is a factor in the causation or propagation of tissue injury in numerous diseases. For encapsulated cell therapy approaches in humans, therefore, (1) unencapsulated cells should be rejected immediately by the host; (2) encapsulated cells should be non-immunogenic to the host; and (3) the cell elimination process should not lead to immunological memory of the host. Accordingly, it would be desirable to be able to deliver a biologically active molecule to human patients using encapsulated cells or tissue that both have a human immunological xe2x80x9cbackground,xe2x80x9d but also provide the safety feature of rejection by the patient in the event of capsule rupture or failure.
The invention provides novel approaches for expressing naturally type I cell surface molecules (i.e., with the carboxy terminus [C-terminus] projecting toward the cytosol and the amino-terminus [N-terminus] projecting away from the cell surface) as type II molecules (i.e., with the N-terminus projecting toward the cytosol and the C-terminus projecting away from the cell surface). The biological function is maintained in the type II orientation. Using this approach (1) cells expressing such molecules can be used as therapeutic agents and (2) a screening process can evaluate the function of novel molecules that were not previously available for testing. The invention also provides a novel approach to predetermining the fate of the transformed cells. Thus, the invention is new aspect of gene therapy and tumor therapy. While gene therapy is a new field, conferring cytotoxic sensitivity on tumor cells has been an area of active research. The strategy described here can be used to target tumor cells; tumor cells expressing immunostimulatory cell surface polypeptides are therefore more susceptible to macrophage clearance.
The invention provides novel immunostimulatory cell surface polypeptides, novel recombinant polynucleotides encoding immunostimulatory cell surface polypeptides, and transformed cells containing the recombinant polynucleotides. When a transformed cell containing a recombinant polynucleotide expresses the encoded immunostimulatory cell surface polypeptide in a host, the host undergoes an immune response that results in rejection of the transformed cell by the host. The host immune response can include the activation of phagocytes, such as macrophages, but does not include complement fixation. In a specific embodiment, the immunostimulatory cell surface polypeptides is a chimeric polypeptide containing the human transferrin receptor membrane domain anchors a human IgG1 Fc to the surface of the cell plasma membrane in a xe2x80x9creversed orientation,xe2x80x9d thus mimicking the configuration of IgG during opsonization. The transformed cells containing the recombinant polynucleotides of the invention are therapeutically useful for the treatment of many disorders.
The invention also provides diagnostic methods for identifying novel therapeutics. In one embodiment, the invention is a method for testing phagocytes for response to an immunostimulatory cell surface polypeptide. A phagocyte is contacted in vitro with a transformed cell containing a recombinant polynucleotide. The recombinant polynucleotide is a promoter operably linked with a polynucleotide coding for an immunostimulatory cell surface polypeptide, and the immunostimulatory cell surface polypeptide activates phagocytes, but does not fix complement. The phagocytic activity of the phagocyte is compared with control phagocyte; and increased phagocytic activity indicates that the phagocyte responds to the immunostimulatory cell surface polypeptide. In another embodiment, the invention is a method for identifying a compound that modulates phagocyte response to an immunostimulatory cell surface polypeptide. A phagocyte is contacted in vitro with a transformed cell containing a recombinant polynucleotide. The process is then repeated by contacting a phagocyte in vitro with a test compound and the transformed cell containing a recombinant polynucleotide. The phagocytic activity of the phagocyte in the absence of the test compound is compared with the phagocytic activity of the phagocyte in the presence of the test compound. A change in the phagocytic activity indicates that the test compound modulates phagocyte response to the immunostimulatory cell surface polypeptide.
The invention further provides a method for stimulating phagocyte activity. A transformed cell containing a recombinant polynucleotide containing a promoter operably linked with a polynucleotide coding for an immunostimulatory cell surface polypeptide is administered to a host. In one embodiment, the stimulated phagocyte is a macrophage, especially a macrophagic tumor cell. In another embodiment, the transformed cell contains a therapeutic compound, such as an anti-tumor compound.
The invention provides a method for modulating an immune response in a host. A transformed cell containing a recombinant polynucleotide with a promoter operably linked with a polynucleotide coding for an immunostimulatory cell surface polypeptide is administered to the host. The administration stimulates an immune response to the transformed cell, because the activation of phagocytes, especially macrophages, acts to regulate both T and B lymphocytes. Macrophages engulf the transformed cell and present the antigenic determinants from the transformed cell to T cells, stimulating an immune response. In one embodiment, the cell expresses, on the cell surface, a xe2x80x9csecond antigen,xe2x80x9d such that the host produces an immune response against the second antigen from the transformed cell. The immunostimulatory cell surface polypeptide enhances the cellular interaction with macrophages. As a result of this enhanced cellular interaction, the second antigen is presented as a target for T-cells. In one embodiment, the transformed cell expresses the second antigen from a recombinant polynucleotide.
The invention provides a method for ablating undesirable target cells, such as tumor cells in a patient, by the targeted delivery of the recombinant polynucleotides of the invention followed by either constitutive or inducible expression of encoded polypeptide. The delivery of the immunostimulatory cell surface polypeptide of the invention into solid tumors results in the selective phagocyte-mediated ablation of the undesirable cells.
The invention provides a method for the treatment of autoimmune disorders in a host, by eliminating autoreactive T-cells. Transformed cells containing a recombinant polynucleotide comprising a promoter operably linked with a polynucleotide coding for an immunostimulatory cell surface polypeptide are administered to a host with an autoimmune disorder. The cells express a therapeutically effective amount of immunostimulatory cell surface polypeptide from the recombinant polypeptide. The immunostimulatory cell surface polypeptide contacting macrophages activates the macrophages to modulate host autoreactive T-cells, thereby reducing the T-cell autoreactivity in the host. Macrophages specifically modulate Th1/Th2 responses. The reactiveness of T-cells differ depending on the availability of co-stimulatory factors. Therefore, T-cells can be induced to become tolerant.
The invention provides a composition in which a transformed cell capable of expressing an immunostimulatory cell surface polypeptide is encapsulated in an immunoisolatory capsule. The transformed cells of the invention are particularly useful when encapsulated for implantation in a human patient, because cells escaping from a ruptured capsule are destroyed by the patient""s immune system. A host immune response will not be triggered by the transformed cells expressing an immunostimulatory cell surface polypeptide in an intact device. In case of a device failure, however, the released cells are effectively eliminated by phagocytes without complement activation or the creation of an immune memory.